Osteoporosis is a disease of progressive bone loss, leading to weak and fracture prone bones. This disease affects ~12 million Americans and is a major medical and economic burden on American society. Osteoporosis is primarily a genetic disorder with fracture predicting traits, such as bone mineral density (BMD), being among the most highly heritable disease associated phenotypes. In humans and mice, genetic studies to date have almost exclusively focused on the analysis of BMD. However, BMD is a complex organismal- level trait that is influenced by a complicated milieu of genetic and environmental factors. This has hampered our ability to precisely identify the causal genes, and more importantly, their mechanisms of action, that underlie genetic associations. As an alternative, we propose to focus exclusively on the genetics of a more 'simple' cell-level process, osteoblast-mediated bone formation. The objective of this proposal is to identify genes affecting osteoblast function. This will be accomplished using a novel and innovative mouse genetic reference population termed the Collaborative Cross (CC). In a pilot study, we identified a genetic locus on mouse Chr 4 for osteoblast-mediated bone formation using genome-wide association mapping and determined that Wnt4 was the candidate gene for this locus. In Aim 1, we will evaluate the effect of modulating expression levels of Wnt4 on osteoblast function in vitro and the effects of deleting Wnt4 on bone mass and strength in vivo. In Aim 2, we will map additional high-resolution quantitative trait loci (QTL) for mineralized nodule formation, a physiologically relevant measure of osteoblast-mediated bone formation, in the CC. In Aim 3, we will move from QTL to the identification of and validation of candidate genes for mineralized nodule formation QTL. This will be accomplished by exploiting the unique genetic aspects of the CC to bioinformatically narrow these loci, followed by RNA-seq/expression QTL studies to further pin point causative genes. Candidate genes for mineralized nodule formation QTL will be tested by gene overexpression and knockdown studies. We expect that the study of a cell-level process will provide the means to more efficiently go from locus to gene to mechanism. Wnt4 and additional genes that will be identified will serve as potential therapeutic targets capable of increasing bone formation in the setting of osteoporosis.